The major objective of this proposal is to study the regulatory mechanisms that control the production of erythropoietin in response to tissue hypoxia. The contribution of transcriptional and post-transcriptional regulation in the expression of the erythropoietin gene in response to hypoxia will be evaluated by "run-on" analysis and by measurement of erythropoietin mRNA half-lives. For this latter purpose cells will be transfected with a plasmid construct which expresses the erythropoietin messenger at very high levels and whose transcriptional activity is independent of hypoxia. The location and limits of cis-acting regulatory regions of the erythropoietin gene will be determined by transient transfection assays in normal and hypoxic erythropoietin producing cells. For these experiments we will use a mini-gene construct which allows the differentiation between the endogenous and the transfected gene. Once the DNA regions containing tissue specific and/or hypoxic specific regulatory elements are determined, the trans-acting factors that interact with these regions will be studied by using DNA-protein interaction techniques and in vitro transcription systems. Attempts will be made to further characterize and purify these trans-acting factors with the use of DNA affinity chromatography. Furthermore, once purification is accomplished, attempts will be made to clone the corresponding genes utilizing expression libraries from erythropoietin producing cells. The elucidation of the mechanisms involved in the regulation of erythropoietin production will increase our understanding of the pathophysiology of anemias and polycythemias.